Plasmodium falciparum-infected human erythrocytes evade host immunity by expression of a cell-surface variant antigen and receptors for adherence to endothelial cells. These properties have been ascribed to P. falciparum erythrocyte membrane protein 1 (PfEMP1), an antigenically diverse malarial protein of 200-350 kDa on the surface of parasitized erythrocytes (PEs). We describe the cloning of two related PfEMP1 genes from the Malayan Camp (MC) parasite strain. Antibodies generated against recombinant protein fragments of the genes were specific for MC strain PfEMP1 protein. These antibodies reacted only with the surface of MC strain PEs and blocked adherence of these cells to CD36 but without effect on adherence to thrombospondin. Multiple forms of the PfEMP1 gene are apparent in MC parasites. The molecular basis for antigenic variation in malaria and adherence of infected erythrocytes to host cells can now be pursued.

We have identified strain-specific antigens with Camp and St. Lucia strains of P. falciparum of Mr approximately 285,000 and approximately 260,000, respectively. These strain-specific antigens were metabolically labeled with radioactive amino acids, indicating that they were of parasite origin rather than altered host components. These proteins had the properties of a molecule exposed on the surface of infected erythrocytes (IE). First, the proteins are accessible to lactoperoxidase-catalyzed radioiodination of IE. Second, the radioiodinated proteins were cleaved by low concentrations of trypsin (0.1 microgram/ml). Third, these antigens were immunoprecipitated after addition of immune sera to intact IE. Fourth, the strain-specific immuno-precipitation of these proteins correlated with the capacity of immune sera to block cytoadherence of IE in a strain-specific fashion. Fifth, the strain-specific antigen had detergent solubility properties (i.e., insolubility in 1% Triton X-100, solubility in 5% sodium dodecyl sulfate) similar to the variant antigen of P. knowlesi, which has been proven to be a malarial protein exposed on the erythrocyte surface.

Adherence of mature Plasmodium falciparum parasitized erythrocytes (PRBCs)

Red blood cells that are infected with the malaria parasite Plasmodium falciparum express new antigens on their surface. In a study of these antigens in the erythrocytes of naturally infected children in the Gambia, an antibody-mediated agglutination assay revealed an extreme degree of antigenic diversity. Serum samples from each of ten children in the convalescent stage of malaria infection reacted with infected cells from the same child but generally not with infected cells from the other children. The Gambian children's erythrocytes also expressed shared determinants: sera from Gambian adults often reacted with the surface of infected cells from all of the children and were shown by adsorption and elution experiments to contain antibodies that recognized several isolates. Conserved determinants exposed on infected erythrocytes may be important for development of antimalarial immunity either naturally or through vaccination.

Abstract. Plasmodium falciparum-infected erythrocytes (IRBCs) synthesize several histidine-rich proteins (HRPs) that accumulate high levels of [3H]histidine but very low levels of amino acids such as [3H]isoleucine or [35S]methionine. We prepared a monoclonal antibody which reacts specifically with one of these HRPs (Pf HRP II) and studied the location and synthesis of this protein during the parasite's intracellular growth.With the knob-positive Malayan Camp strain of P. falciparum, the monoclonal antibody identified a multiplet of protein bands with major species at Mr 72,000 and 69,000. Pf HRP II synthesis began with immature parasites (rings) and continued through the trophozoite stage. The Mr 72,000 band of Pf HRP II, but not the faster moving bands of the multiplet, was recovered as a water-soluble protein from the culture supernatant of intact IRBCs. Approximately 50% of the total [3H]histidine radioactivity incorporated into the Mr 72,000 band was extracellular between 2 and 24 h of culture. Immunofluorescence and cryothin-section immunoelectron microscopy localized Pf HRP II to several cell compartments including the parasite cytoplasm, as concentrated "packets" in the host erythrocyte cytoplasm and at the IRBC membrane. Our results provide evidence for an intracellular route of transport for a secreted malarial protein from the parasite through several membranes and the host cell cytoplasm.T HE avian malaria Plasmodium lophurae and the major pathogenic human malaria P. falciparum synthesize proteins of extraordinary histidine content called histidine-rich proteins (HRPs). I Asexual blood stage P. lophurae parasites synthesize an HRP (P1 HRP) containing 72 mol % histidine according to amino acid analysis (8) and sequencing of genomic DNA (17). Despite the fact that in duckling infections it can account for 10% of the total parasite dry weight (8) its function is unknown. At least three HRPs are made by blood stage P. falciparum, denoted here as Pf HRP I, II, and III in order of discovery. Pf HRP I is associated phenotypically with expression of knob-like protrusions on the surface membrane of infected erythrocytes (IRBCs) (9). Pf HRP I appears to be associated with the IRBC membrane (23), particularly the cytoskeleton (13). Pf HRP II, described in detail in this report, is expressed by both knob-positive (K ÷) and knob-negative (K-) IRBCs (13), unlike Pf HRP I which is only expressed by K ÷ IRBC (5, 9, 24). Sequencing of genomic DNA has shown that Pf HRP II contains 35% histidine, as well as relatively high contents of alanine and aspartate (40 and 12 %, respectively) (25). Pf HRP III has been recently identified by sequencing a cDNA clone and shown to contain 30% histidine and 29% alanine (19).Since the roles that these HRPs play in the complex interaction of blood-stage malaria parasite and vertebrate host are unknown, and the structure-function relationship of proteins with such high contents of imidazole groups are still a matter for conjecture, we have prepared monoclonal antibodies (mAb's) to diss...